Structure Of Cytochrome C Oxidase Research Articles

Resonance Raman spectral analysis of the heme site structure of cytochrome c oxidase with its positive regulator CHCHD2

Cytochrome c oxidase (CcO) reduces O2, pumps protons in the mitochondrial respiratory chain, and is essential for oxygen consumption in the cell. The coiled-coil-helix-coiled-coil-helix domain-containing 2 (CHCHD2; also known as mitochondrial nuclear retrograde regulator 1 [MNRR1], Parkinson's disease 22 [PARK22] and aging-associated gene 10 protein [AAG10]) is a protein that binds to CcO from the intermembrane space and positively regulates the activity of CcO. Despite the importance of CHCHD2 in mitochondrial function, the mechanism of action of CHCHD2 and structural information regarding its binding to CcO remain unknown. Here, we utilized visible resonance Raman spectroscopy to investigate the structural changes around the hemes in CcO in the reduced and CO-bound states upon CHCHD2 binding. We found that CHCHD2 has a significant impact on the structure of CcO in the reduced state. Mapping of the heme peripheries that result in Raman spectral changes in the structure of CcO highlighted helices IX and X near the hemes as sites where CHCHD2 takes action. Part of helix IX is exposed in the intermembrane space, whereas helix X, located between both hemes, may play a key role in proton uptake to a proton-loading site in the reduced state for proton pumping. Taken together, our results suggested that CHCHD2 binds near helix IX and induces a structural change in helix X, accelerating proton uptake.

Direct Interaction of Mitochondrial Cytochrome c Oxidase with Thyroid Hormones: Evidence for Two Binding Sites.

Thyroid hormones regulate tissue metabolism to establish an energy balance in the cell, in particular, by affecting oxidative phosphorylation. Their long-term impact is mainly associated with changes in gene expression, while the short-term effects may differ in their mechanisms. Our work was devoted to studying the short-term effects of hormones T2, T3 and T4 on mitochondrial cytochrome c oxidase (CcO) mediated by direct contact with the enzyme. The data obtained indicate the existence of two separate sites of CcO interaction with thyroid hormones, differing in their location, affinity and specificity to hormone binding. First, we show that T3 and T4 but not T2 inhibit the oxidase activity of CcO in solution and on membrane preparations with Ki ≈ 100–200 μM. In solution, T3 and T4 compete in a 1:1 ratio with the detergent dodecyl-maltoside to bind to the enzyme. The peroxidase and catalase partial activities of CcO are not sensitive to hormones, but electron transfer from heme a to the oxidized binuclear center is affected. We believe that T3 and T4 could be ligands of the bile acid-binding site found in the 3D structure of CcO by Ferguson-Miller’s group, and hormone-induced inhibition is associated with dysfunction of the K-proton channel. A possible role of this interaction in the physiological regulation of the enzyme is discussed. Second, we find that T2, T3, and T4 inhibit superoxide generation by oxidized CcO in the presence of excess H2O2. Inhibition is characterized by Ki values of 0.3–5 μM and apparently affects the formation of O2●− at the protein surface. The second binding site for thyroid hormones presumably coincides with the point of tight T2 binding on the Va subunit described in the literature.

Structure of bovine cytochrome c oxidase in the ligand-free reduced state at neutral pH.

Cytochrome c oxidase (CcO), the terminal oxidase in cellular respiration, couples proton pumping to O2 reduction. Mammalian CcO resides in the inner mitochondrial membrane. Previously, a model of H-pathway proton pumping was proposed based on various CcO crystal structures. However, all previously determined structures were solved using crystals obtained at pH 5.7, which differs from the environmental pH of CcO in the inner membrane. The structures of fully oxidized and ligand-free reduced CcO at pH 7.3 have now been determined. Structural comparison between the oxidized and reduced states revealed that the structural alterations that occurred upon redox change at pH 5.7 in Asp51, the magnesium-containing cluster, haem groups and helix X, which provide important structural evidence for the H-pathway proton-pumping proposal, also occur at pH 7.3. These structural alterations were restricted to a local region of CcO; no domain movement was detected, nor were significant structural alterations detected in peripheral regions at either pH value. These observations indicate that the small and precise structural alterations that occur over the course of the reaction cycle are not affected by pH change, and that isolated CcO precisely performs proton pumping via the H-pathway over a wide pH range. Because the pH is not uniform across the molecular surface of CcO, the fact that the overall structure of CcO is not affected by pH changes ensures the high enzymatic efficiency of this protein in the mitochondria.

Sequential Dissociation of Subunits from Bovine Heart Cytochrome c Oxidase by Urea

The quaternary stability of purified, detergent-solubilized, cytochrome c oxidase (CcO) was probed using two chemical denaturants, urea and guanidinium chloride (GdmCl). Each chaotrope induces dissociation of five subunits in a concentration-dependent manner. These five subunits are not scattered over the surface of CcO but are clustered together in close contact at the dimer interface. Increasing the concentration of urea selectively dissociates subunits from CcO in the following order: VIa and VIb, followed by III and VIIa, and finally Vb. After incubation in urea for 10 min at room temperature, the sigmoidal dissociation transitions were centered at 3.7, 4.6, and 7.0 M urea, respectively. The secondary structure of CcO was only minimally perturbed, indicating that urea causes disruption of subunit interactions without urea-induced conformational changes. Incubation of CcO in urea for 120 min produced similar results but shifted the sigmoidal dissociation curves to lower urea concentrations. Incubation of CcO with increasing concentrations of GdmCl produces an analogous effect; however, the GdmCl-induced dissociation of subunits occurs at lower concentrations and with a narrower concentration range. Thermodynamic parameters for each subunit dissociation were evaluated from the sigmoidal dissociation data by assuming a single transition from bound to dissociated subunit. The free energy change accompanying urea-induced dissociation of each subunit ranged from 18.0 to 29.7 kJ/mol, which corresponds to 0.32-0.59 kJ/mol per 100 A(2) of newly exposed solvent-accessible surface area. These values are 30-50-fold smaller than previously reported for the unfolding of soluble or membrane proteins.

Structure of cytochromec oxidase from baker's yeast—a progress report. Preparation of four subunits for amino acid sequence determination and attempts to localize the cytochromec binding site

Cytochrome c oxidase from the inner membrane of yeast mitochondria consists of seven nonidentical protein subunits, three being synthesized on mitochondrial ribosomes (molecular weights I: 43 K, II: 34 K, and III: 24 K) and four being made on cytoplasmic ribosomes (molecular weights IV: 14 K, V: 12 K, VI: 12 K, and VII: 4.5 K). In the present study all four cytoplasmically synthesized subunits of the enzyme were isolated on a large scale using ion exchange chromatography and gel filtration. Their amino acid composition as well as their amino- and carbosy-terminal amino acid residues have been determined. Sequence determinations of subunits IV and VI are already in an advanced state. The sequence of subunit VI is characterized by a large amino-terminal stretch dominated by charged amino acid residues followed by a cluster of hydrophobic amino acids. The binding site of yeast cytochrome oxidase for cytochrome c was studied by chemical crosslinking experiments. The formation of a disulfide bridge between the two proteins was observed by using cytochrome c from yeast modified with 5-thionitrobenzoate at the cysteinyl residue in position 107. Alternatively, a disulfide between yeast cytochrome c and the oxidase could be formed directly by oxidation with copper phenanthroline. Gel electrophoresis of the crosslinked complexes in sodium dodecyl sulfate revealed a new protein band with an apparent molecular weight of 38 K. This new band appears to be derived from cytochrome c and from subunit III of cytochrome oxidase.